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Death and Baxes: Mechanisms of lymphotrophic cytokines
Abstract
In this review, we briefly cover the critical requirements for interleukin-7 (IL-7) in thymocyte development and peripheral T-cell homeostasis. Part of the IL-7 effect is antiapoptotic or 'trophic' and we have studied the intracellular pathways involved in lymphocyte survival and death regulated by this cytokine. We review the evidence for a role of the JAK signal transducers and activators of transcription protein (STAT) pathway and phosphoinositide 3-kinase (PI3K)-AKT pathways in survival. The death pathway following IL-7 withdrawal is discussed in terms of the balance of BCL-2 vs. BAX and other death proteins and the role of metabolic disturbances involving glucose metabolism and intracellular pH. The IL-7 survival and death pathways in lymphocytes may be representative of many trophic factors in different cell types; yet we conclude that much of the mechanism remains to be discovered.
... There exists a number of IL-7 functional targets [31]. To name a few, IL-7 promotes cell survival through Bcl-2 upregulation and repression of Bax323334, Bim [35] or Bad [36]. IL-7 drives proliferation through cell cycle regulators such as Cdc25A [19] and p27 kip [37]. ...
... IL-7 drives proliferation through cell cycle regulators such as Cdc25A [19] and p27 kip [37]. IL-7 also regulates glucose metabolism [34,38], and this function is partially fulfilled through transcriptional control of Hexokinase II (HXKII) [39]. How other signaling pathways augment the JAK/STAT signal and contribute to T-cell growth is unknown. ...
Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting immune reconstitution. This feature makes IL-7 an ideal candidate for therapeutic development. As with other cytokines, signaling through the IL-7 receptor induces the JAK/STAT pathway. However, the broad scope of IL-7 regulatory targets likely necessitates the use of other signaling components whose identities remain poorly defined. To this end, we used an IL-7 dependent T-cell line to examine how expression of the glycolytic enzyme, Hexokinase II (HXKII) was regulated by IL-7 in a STAT5-independent manner. Our studies revealed that IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Gel shifts showed that the AP-1 complex induced by IL-7 contained JunD but not c-Fos or c-Jun. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. Because others had shown that JunD was a negative regulator of cell growth, we performed a bioinformatics analysis to uncover possible JunD-regulated gene targets. Our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism. One of these growth promoters was the oncogene, Pim-1. Pim-1 is an IL-7-induced protein that was inhibited when the activities of JNK or JunD were blocked, showing that in IL-7 dependent T-cells JunD can promote positive signals transduced through Pim-1. This was confirmed when the IL-7-induced proliferation of CD8 T-cells was impaired upon JunD inhibition. These results show that engagement of the IL-7 receptor drives a signal that is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth stimulation through the expression of metabolic and signaling factors like HXKII and Pim-1.
... Mediators in the intrinsic apoptotic pathway are the major effectors downstream of IL-7 signaling. It is well-known that IL-7 activates a Jak/STAT signaling pathway [218]. STAT5 activates Bcl-2 expression and a Bcl-2 transgene can rescue the defect in IL-7R -/mice [9,10]. ...
... Similarly, a Bcl-2 transgene or a deficiency in Bax partially rescues the severe defects in lymphopoiesis in mice lacking Jak3, which is downstream of IL-7 signaling [219]. Furthermore, IL-7 signal activates PI-3K/Akt [218]. It is well established that the PI-3K/Akt pathway phosphorylates the pro-apoptotic protein Bad, which inhibits its function [68,220]. ...
Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work together to regulate T lymphocyte development and function.
... in septic patients treated with CYT107 is the potent antiapoptotic effect of IL-7 (25)(26)(27)(28)(29). IL-7 increases antiapoptotic Bcl-2 family members and decreases proapoptotic Bcl-2 family members (53). Previously, our laboratory utilized a mouse peritonitis model of sepsis to demonstrate that CYT107 decreased apoptosis of splenic and lymph node naive, central memory, and effector memory CD4 + and CD8 + T cells by approximately 50% at 24 hours (26). ...
Background:
A defining pathophysiologic feature of sepsis is profound apoptosis-induced death and depletion of CD4+ and CD8+ T cells. Interleukin-7 (IL-7) is an antiapoptotic common γ-chain cytokine that is essential for lymphocyte proliferation and survival. Clinical trials of IL-7 in over 390 oncologic and lymphopenic patients showed that IL-7 was safe, invariably increased CD4+ and CD8+ lymphocyte counts, and improved immunity.
Methods:
We conducted a prospective, randomized, double-blind, placebo-controlled trial of recombinant human IL-7 (CYT107) in patients with septic shock and severe lymphopenia. Twenty-seven patients at academic sites in France and the United States received CYT107 or placebo for 4 weeks. Primary aims were to determine the safety of CYT107 in sepsis and its ability to reverse lymphopenia.
Results:
CYT107 was well tolerated without evidence of inducing cytokine storm or worsening inflammation or organ dysfunction. CYT107 caused a 3- to 4-fold increase in absolute lymphocyte counts and in circulating CD4+ and CD8+ T cells that persisted for weeks after drug administration. CYT107 also increased T cell proliferation and activation.
Conclusions:
This is the first trial of an immunoadjuvant therapy targeting defects in adaptive immunity in patients with sepsis. CYT107 reversed the marked loss of CD4+ and CD8+ immune effector cells, a hallmark of sepsis and a likely key mechanism in its morbidity and mortality. CYT107 represents a potential new way forward in the treatment of patients with sepsis by restoring adaptive immunity. Such immune-based therapy should be broadly protective against diverse pathogens including multidrug resistant bacteria that preferentially target patients with impaired immunity.
Trial registration:
Trials registered at clinicaltrials.gov: NCT02640807 and NCT02797431.
Funding:
Revimmune, NIH National Institute of General Medical Sciences GM44118.
... Bcl-2) and decrease pro-apoptotic protein expression (e.g. Bad and Bax) [1][2][3][4][5]. Given its role in T-cell homeostasis, IL-7 immunotherapy has been widely applied to diseases in which T-cells may play a role. ...
The use of interleukin-7 (IL-7) as an immunorestorative therapeutic has proven effective in HIV infection, cancer and bone marrow transplantation. Mediating its activity through membrane-bound IL-7 receptor α (mCD127), IL-7 therapy increases T-cell numbers and survival. A soluble form, sCD127, is found in plasma, and we have previously identified increased plasma sCD127 concentrations in HIV infection. Furthermore, patients with high sCD127 exhibited the best viral control, implicating a role for IL-7 or sCD127 directly in improved virologic/immunologic outcomes. The role of the cytokine IL-7 in elevating sCD127 levels was addressed here through assessment of retrospective samples obtained from SIV-infected antiretroviral (ART)-treated Rhesus macaques. IL-7 was administered in clustered weekly doses, allowing for an assessment prior, during and following IL-7 administration. The levels of sCD127 remained relatively unchanged during both early SIV infection and following initiation of ART. However, treatment with IL-7 increased sCD127 concentrations in most animals, transiently or persistently, paralleling increased T-cell numbers, correlating significantly with CD8⁺ T-cell levels. In addition, proliferating CD4⁺ or CD8⁺ T-cells (measured by Ki67) increased in association with elevated sCD127 concentrations. Finally, a high concentration of sCD127 in IL7-treated animals was associated with increased retention of T-cells (measured by BrDU). In addition, a lack, or loss of viral control was associated with more pronounced and frequent elevations in plasma sCD127 concentrations with IL-7 therapy. In summary, plasma sCD127 levels in SIV-infected ART-treated macaques was associated with therapeutic IL-7 administration, with higher sCD127 levels in macaques demonstrating the best T-cell responses. This study furthers our knowledge regarding the interrelationship between increased IL-7 levels and elevated sCD127 levels that may have implications for future IL-7 immunotherapeutic approaches in HIV-infected patients.
... Suppression of pro-apoptotic proteins is another way by which IL-7 improves the survival of lymphocytes [1]. Apart from the anti-apoptotic activities, IL-7 regulate the metabolism and pH in T-cells [8,9]. The most important function of IL-7 is the production and homeostatic maintenance of memory T-cells [10]. ...
Aim:
Interleukin 7 (IL-7) is a ϒc family cytokine involved in the homeostatic proliferation and maintenance of immune cells. In the present study, we report the expression of bovine IL-7 (bIL-7) in Escherichia coli and evaluated for its biological activity.
Materials and methods:
The sequence coding for bIL-7 (mature protein) was amplified from primary bovine kidney cell culture and cloned into pET28-a vector and expressed in E. coli (BL 21 DE3). The expressed protein was purified by nickel-nitrilotriacetatechromatography, and the reactivity of the protein was confirmed by western blotting using monoclonal antibodies raised against human IL-7. The biological activity of expressed bIL-7 was evaluated by analyzing its effect on the expression of anuclear factor for activated T-cells c1 (NFATc1), B-cell lymphoma 2 (Bcl2), suppressor of cytokine signaling 3 (SOCS3) molecules in bovine peripheral blood mononuclear cells (PBMCs) by quantitative polymerase chain reaction. Ability of the expressed protein was also analyzed by its effect on phosphorylating signal transducer and activator 3 (STAT3) molecule by immunostaining in human embryonic kidney cells 293 (HEK293) cells.
Results:
The bIL-7 was able to induce the expression of Bcl2 and NFATc1expression in bovine PBMCs by 7 and 5-folds, respectively, whereas a 2-fold decrease was observed in the case of SOCS3 expression. Immunostaining studies in HEK293 cells using antihuman phospho-STAT3 showed activation and nuclear translocation of STAT3 molecule on bIL-7 treatment.
Conclusion:
bIL-7 gene was successfully amplified, cloned, and expressed in a prokaryotic expression system. The biological activity study showed that the E. coli expressed bIL-7 protein is biologically active. Considering the role of IL-7 in T-cell homeostasis and memory cell generation, this molecule can be used for enhancing the vaccine response and that has to be proved by further experiments.
... The functional roles for some of these cytokines will be described more extensively in later sections. Since IL-7 is important in the homeostasis of the T cell compartment, its increased production following HIV infection has been proposed to accelerate thymic output to counter the CD4 T cell loss [105,116,117]. CD8 T cells not only succumbed to cell death through IL-7-induced Fas-mediated apoptosis, but also suffered a loss of their cytolytic activity as well as a decline in CD8 expression and increased IL-4 production [112,[118][119][120]. ...
... This heterogenous association is essential for the biological effect of IL-7. The signaling pathways induced by the binding of IL-7 to its receptor have been well studied and involve activation of the JAK/STAT pathway as well as the PI3-kinase pathway which all lead to increased expression of genes that promote lymphocyte survival and reduced expression of genes that induce lymphocyte apoptosis and cell cycle arrest (Benczik and Gaffen, 2004), (Khaled and Durum, 2003). ...
... Correspondingly, IL-7 cytokine dependent T cell lines, when withdrawn from IL-7, decrease glucose uptake (Khaled and Durum, 2003). Similarly, primary T cells experience a decrease in glucose uptake in vitro, and this reduction can be partially rescued by IL-7 . ...
... Cytokines comprise a large family of extracellular and soluble ligands that form an integrated network of cell-cell communication and humoral interactions regulating cell growth, differentiation, and survival (1). An understanding of the roles played by cytokines in growth and survival has been developed, in part, by studies of murine bone marrow-derived IL-3-dependent hematopoietic cell lines such as Ba/F3, FDC-P1, and FL.5 (2)(3)(4). These lines respond to stimulation with IL-3 as well as with GM-CSF and M-CSF. ...
... IL-7 maintains metabolic activity through the uptake of glucose from the extracellular milieu. In IL-7 dependent cell lines, deprivation of IL-7 resulted that cells rapidly stop glucose transport (Khaled and Durum 2003). The main glucose transporter in peripheral T-cells is Glut1, which is also the receptor for the human T-cell leukemia virus (HTLV) (Manel, Kim et al. 2003). ...
... 7 The IL-7R complex consists of 2 chains, the high-affinity IL-7R␣ chain and ␥c. 9 IL-7 binding to the receptor induces sequential phosphorylation of Jak1, Jak3, and STAT5, leading to the upregulation of Bcl-2, which promotes cell survival. 10,11 IL-7 regulates peripheral homeostasis of CD4 ϩ and CD8 ϩ T cells by promoting cell survival. When CD8 ϩ T cells from IL-7R intact and knockout (KO) mice were adoptively transferred to wild-type mice, cells from IL-7R KO mice had decreased survival compared with those from IL-7R intact mice. ...
... The choice of using RT activity as a measure of viral latency was dictated by the evidence that cell-associated viral RNA can be detected in latently infected resting memory CD4 ϩ T cells of HIV-1-infected subjects under effective antiretroviral therapy (11,51), and therefore, this parameter might not provide a stringent measure of latency. This result underscores the role of low doses of IL-7 in the establishment of immunological memory in CD4 ϩ T cells (52)(53)(54). We have previously shown that the HIV-1 antigen p24 gag produced during active viral replication persists in the cytoplasm of infected cells for several days, even during latent infection, which allows us to distinguish latently infected CD4 ϩ T cells from their uninfected counterparts (20). ...
Resting memory CD4+ T cells are the largest reservoir of persistent infection in HIV-1-positive subjects. They harbor dormant, stably integrated
virus despite suppressive antiretroviral therapy, posing an obstacle to a cure. Surface markers that identify latently infected
cells remain unknown. Microarray analyses comparing resting latently infected and uninfected CD4+ T cells generated in vitro showed profound differences in the expression of gene programs related to transcriptional and posttranscriptional regulation,
cell proliferation, survival, cycle progression, and basic metabolism, suggesting that multiple biochemical and metabolic
blocks contribute to preventing viral production in latently infected cells. We identified 33 transcripts encoding cell surface
markers that are differentially expressed between latently infected and uninfected cells. Quantitative reverse transcriptase
PCR (RT-QPCR) and flow cytometry analyses confirmed that the surface marker CD2 was expressed at higher levels on latently
infected cells. To validate this result in vivo, we sorted resting memory CD4+ T cells expressing high and low surface levels of CD2 from six HIV-1-infected subjects successfully treated with antiretroviral
drugs for at least 3 years. Resting memory CD4+ CD2high T cells from all subjects harbored higher HIV-1 DNA copy numbers than all other CD4+ T cell subsets. Moreover, after ex vivo viral reactivation, robust viral RNA production was detected only from resting memory CD4+ CD2high T cells but not from other cell subsets. Altogether, these results show that a high CD2 expression level is a hallmark of
latently infected resting memory CD4+ T cells in vivo.
... Accordingly, in these stages of cell development, different survival signals, such as IL7 or BCR tonic signals trigger elevated levels of A1 or Mcl-1 anti-apoptotic proteins of the Bcl-2 family (Opferman, 2008). However, upon antigen recognition, immunoreceptor triggering, cytokines or growth factors (mainly through the regulation of AKT; Suzuki et al., 2003), the Jak-STAT (Khaled and Durum, 2003) pathway, and elevated metabolism (Khan, 2009) strongly push the balance of pro-versus anti-apoptotic members of Bcl-2 family toward survival. Therefore activated lymphocytes must be relatively resistant to the intrinsic apoptotic pathway. ...
Cell death receptors have crucial roles in the regulation of immune responses. Here we review recent in vivo data confirming that the Fas death receptor (TNFSR6) on B cells is important for the regulation of autoimmunity since the impairment of only Fas function on B cells results in uncontrolled autoantibody production and autoimmunity. Fas plays a role in the elimination of the non-specific and autoreactive B cells in germinal center, while during the selection of antigen-specific B cells different escape signals ensure the resistance to Fas-mediated apoptosis. Antigen-specific survival such as BCR or MHCII signal or coreceptors (CD19) cooperating with BCR inhibits the formation of death inducing signaling complex. Antigen-specific survival can be reinforced by antigen-independent signals of IL-4 or CD40 overproducing the anti-apoptotic members of the Bcl-2 family proteins.
... To this end, we generated the SMoR T-cell line from BimKO mice as described in Materials and Methods. To demonstrate that the expression of Bcl-2 family members in D1 or SMoR cells was not altered by either the immortalization process or loss of p53 or Bim, we examined Bcl-2 and Bax, proteins whose expression or activity, respectively, is regulated by IL-7 [16,37,50]. As shown in Figure 2A, we found that the two cell lines displayed minimal differences in the amounts of Bcl-2 or Bax detected in response to IL-7. ...
Bim is a BH3-only member of the Bcl-2 family that enables the death of T-cells. Partial rescue of cytokine-deprived T-cells occurs when Bim and the receptor for the T-cell growth factor, interleukin-7, are deleted, implicating Bim as a possible target of interleukin-7-mediated signaling. Alternative splicing yields three major isoforms: BimEL, BimL and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on interleukin-7 for growth, were used. Loss of total Bim in interleukin-7-deprived T-cells resulted in delayed apoptosis. However, loss of Bim also impeded the later degradative phase of autophagy. p62, an autophagy-adaptor protein which is normally degraded, accumulated in Bim deficient cells. To explain this, BimL was found to support acidification of lysosomes that later may associate with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon interleukin-7 withdrawal only in Bim-containing T-cells. intereukin-7 dependent T-cells lacking Bim were less sensitive to inhibition of lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, promoted intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of degradative autolysosomes.
... Some evidence also suggests that Atg5 interacts with FADD, an important regulator of the extrinsic pathway of apoptosis (16). In addition to these direct interactions, autophagy and the Bcl-2 family can both be regulated by the Akt and mTOR pathways in T lymphocytes (98,121). The balance of these types of intracellular signals will likely determine whether a cell survives or dies and through which mechanism. ...
During the development and normal function of T lymphocytes, the cells are subject to several checkpoints at which they must "decide" to live or die. At these critical times and during homeostasis, the molecules that regulate the classical apoptotic pathways and survival pathways such as autophagy have critical roles in controlling this decision. Our laboratory has focused on the roles of apoptotic and autophagic proteins in T lymphocyte development and function. Using genetic models in mice and in vitro analyses of T cell functions, we have outlined critical roles for the Bcl-2 family (regulators of the intrinsic pathway of apoptosis), c-FLIP (an anti-apoptotic protein in the extrinsic pathway of apoptosis), and autophagy in T lymphocytes.
... The up-regulation of Bcl-2 pro-survival proteins is thought to counteract the activation of the pro-apoptotic Bcl-2 proteins such as Bim and Bax, which are both essential for cytokine deprivation-induced apoptosis. [18][19][20][21] Cells that express low levels of IL-7R were shown to be susceptible to Bim-induced apoptosis in vivo, whereas those expressing higher levels of IL-7R are protected, 22,23 and Bax deficiency partially compensates IL-7R deficiency. 9 Furthermore, IL-7 has also been shown to activate cell survival signalling pathways including the Janus kinase/signal transducer and activator of transcription (JAK/STAT) and the phosphatidylinositol 3-kinase (PI3K)/ AKT (also referred as protein kinase B) pathways. ...
SUMMARY: Interleukin-7 (IL-7) is a crucial cytokine involved in T-cell survival and development but its signalling in human T cells, particularly in effector/memory T cells, is poorly documented. In this study, we found that IL-7 protects human CD4(+) effector/memory T cells from apoptosis induced upon the absence of stimulation and cytokines. We show that IL-7 up-regulates not only Bcl-2 but also Bcl-xL and Mcl-1 as well. Interleukin-7-induced activation of the janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway is sufficient for cell survival and up-regulation of Bcl-2 proteins. In contrast to previous studies with naive T cells, we found that IL-7 is a weak activator of the phosphatidylinositol 3 kinase (PI3K)/AKT (also referred as protein kinase B) pathway and IL-7-mediated cell survival occurs independently from the PI3K/AKT pathway as well as from activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Considering the contribution of both IL-7 and CD4(+) effector/memory T cells to the pathogenesis of autoimmune diseases such as rheumatoid arthritis and colitis, our study suggests that IL-7 can contribute to these diseases by promoting cell survival. A further understanding of the mechanisms of IL-7 signalling in effector/memory T cells associated with autoimmune inflammatory diseases may lead to potential new therapeutic avenues.
... However, IL-7 also reactivates latent HIV-1 (12,32), and thus using IL-7 to promote the establishment of latency in vitro has been difficult. The antiapoptotic protein Bcl-2 is a downstream effector of IL-7 signaling and plays an essential role in counteracting the proapoptotic effects of cytokine withdrawal and maintaining the survival of resting memory CD4 + T cells (29,33,34). The prosurvival effect via Bcl-2 is the key homeostatic function of IL-7. ...
The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-kappaB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.
... Interleukin (IL)-7 plays an important role in the generation and maintenance of memory T cells, rescuing them from apoptosis, promoting homeostatic self-renewal [3][4][5], up regulating anti-apoptotic factor expression [3,[6][7][8] and inactivating pro-apoptotic proteins [9]. The IL-7 receptor a (IL-7Ra or CD127) is needed to trigger the memory pathway, and several studies suggest that IL-7Ra is a marker of memory cells. ...
HIV-specific CD8+ T cells from patients with primary HIV infection (PHI) and after antiretroviral therapy initiation were evaluated for CD127 expression and proliferative capacity and were compared with cells from chronically-infected patients, including long-term nonprogressors and HIV controllers.
We studied 30 patients with PHI (from the Agence Nationale de Recherche sur le SIDA Primo-infection Cohort) and 33 patients with chronic HIV infection (including nonprogressor patients from the Agence Nationale de Recherche sur le SIDA ALT Cohort and the Agence Nationale de Recherche sur le SIDA HIV Controllers Study Group). HIV-specific CD8+ T cells were identified by costaining with HIV human leukocyte antigen class I pentamers. CD127 expression was assessed by flow cytometry and cell proliferation by carboxyfluorescein succinimidyl ester labeling.
During PHI, most HIV-specific CD8+ T cells coexpressed CD27 and CD45RO, were highly activated, and showed weak Bcl-2 expression. Their CD127 expression was very low and correlated negatively both with HIV RNA and DNA levels and with expression of the activation marker CD38. CD127 expression correlated positively with CD4 cell count, Bcl-2 expression and proliferative capacity. Strong CD127 expression was observed in the two groups of chronically-infected nonprogressors. CD127 expression on HIV-specific CD8+ T cells increased in early-treated PHI patients, reaching levels similar to those observed in nonprogressors. In parallel, these cells acquired strong proliferative capacity. No change in CD127 expression or proliferative potential was observed in untreated patients.
Early antiretroviral therapy initiation enhances CD127 expression on HIV-specific CD8+ T cells, reaching levels similar to those observed in aviremic nonprogressors, and restores their proliferative capacity.
... As an immunotherapeutic agent, IL-7 can augment T cell numbers by 1) increasing thymic output (Henson et al., 2004), 2) promoting T-cell survival (Kim et al., 1998;Khaled and Durum, 2003), and 3) inducing cell cycling (Geiselhart et al., 2001;Kittipatarin et al., 2006). While the activity of IL-7 as a survival factor promoting thymic development has been well documented (Maraskovsky et al., 1997;Khaled et al., 1999), its function as a proliferative factor remains poorly understood. ...
Interleukin-7 (IL-7) increases lymphocyte numbers, a critical feature of immune reconstitution, through mechanisms that are still poorly understood. Part of the problem is that IL-7 is produced in limited amounts by non-lymphoid cells, making in vivo studies of the cytokine's activity a challenge. To overcome this, we developed an in vitro system by which lymphocytes from secondary immune organs could be cultured to produce IL-7 responsive cells. Using this method, we showed that CD8(hi)CD44(hi) T cells accumulate in culture with IL-7 from a population of lymph node or splenic cells. These results were validated when a similar lymphocyte subset was found in mice expressing a constitutively active form of STAT5b, a key transducer of IL-7 signals. Interestingly, IL-7-expanded cells also up regulated the activation marker, CD69. The IL-7-derived CD44(hi)CD69(hi) cells were not generated from naïve cells, but expanded from an existing population, since culture in IL-7 of naïve lymphocytes from OT-1/Rag1(-/-) mice did not produce CD44(hi)CD69(hi) cells. Using the in vitro culture system to study lymphocytes from mice deficient in the apoptotic protein, BIM, we were able to attribute the expansion of CD8(hi)CD44(hi)CD69(hi) T cells to the proliferative and not survival activity of IL-7. The in vitro culture system provides an important new methodology to examine the activities of this essential as well as immunotherapeutic cytokine.
... Cytokines comprise a large family of extracellular and soluble ligands that form an integrated network of cell-cell communication and humoral interactions regulating cell growth, differentiation, and survival (1). An understanding of the roles played by cytokines in growth and survival has been developed, in part, by studies of murine bone marrow-derived IL-3-dependent hematopoietic cell lines such as Ba/F3, FDC-P1, and FL.5 (2)(3)(4). These lines respond to stimulation with IL-3 as well as with GM-CSF and M-CSF. ...
IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3gamma was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3gamma overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3gamma in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3gamma, resulting in cell death. These results indicate that deregulated expression of 14-3-3gamma may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.
... Among factors that activate NF-kB/Rel in leukocytes, cytokines, including interleukin (IL)-1, -2, GM-CSF, tumor necrosis factors and others mediate paracrine signals originating from the cell environment (Li and Verma, 2002). IL-7, produced by stromal cells from bone marrow, thymus and other tissues, is a survival factor of key relevance for the differentiation and survival of lymphocytes (Alpdogan et al., 2003;Jaleco et al., 2003;Khaled and Durum, 2003;Napolitano et al., 2003). Here we analyzed (A) Dose-response analysis of NF-kB/Rel activation by IL-7. ...
Pathways that regulate the activation of NF-kappaB/Rel transcription factors are known to include signaling through a number of cytokine receptors. Interleukin 7 (IL-7), produced by bone marrow and other stromal cells, is a key factor for differentiation and survival in the lymphoid and other compartments. We found that human recombinant IL-7 induced NF-kappaB/Rel activation, analyzed by electrophoretic mobility shift assay (EMSA), in human peripheral blood T lymphocytes from healthy donors. Induced complexes included p65 and p50 NF-kappaB/Rel subunits. These results demonstrate for the first time that IL-7 can participate in signaling leading to NF-kappaB/Rel activation.
... The γ c cytokine IL-7 and interactions between the TCR and self-peptide/MHC maintain T cell viability, allow homeostatic proliferation in vivo and can maintain basal T cell size and metabolism in vitro [3,5,8,9]. The role of GPCR in these T cell homeostatic processes, however, is not known. ...
Signals that regulate T cell homeostasis are not fully understood. G protein-coupled receptors (GPCR), such as the chemokine receptors, may affect homeostasis by direct signaling or by guiding T cell migration to distinct location-restricted signals. Here, we show that blockade of Galphai-associated GPCR signaling by treatment with pertussis toxin led to T cell atrophy and shortened life-span in T cell-replete hosts and prevented T cell homeostatic growth and proliferation in T cell-deficient hosts. In vitro, however, neither GPCR inhibition nor chemokine stimulation affected T cell atrophy, survival, or proliferation. These findings suggest that GPCR signals are not trophic stimuli, but instead may be required for migration to distinct trophic signals, such as IL-7 or self-peptide/MHC. Surprisingly, while chemokines did not affect atrophy, atrophic T cells displayed increased chemokine-induced chemotaxis that was prevented by IL-7 and submitogenic anti-CD3 antibody treatment. This increase in migration was associated with increased levels of GTP-bound Rac and the ability to remodel actin. These data suggest a novel mechanism of T cell homeostasis wherein GPCR may promote T cell migration to distinct location-restricted homeostatic trophic cues for T cell survival and growth. Homeostatic trophic signals, in turn, may suppress chemokine sensitivity and cytoskeletal remodeling, to inhibit further migration.
... It is known that the PI3-K/Akt signaling pathway transmits survival or antiapoptotic signaling and is activated by stimulation through several cytokine receptors [28]. Because eNOS activation is downstream of Akt phosphorylation, eNOS-mediated production of NO may provide a survival signal and is consistent with other reports that NO can be antiapoptotic [6][7][8]10,11]. ...
Oxidative stress mediates positive and negative effects on physiological processes. Recent reports show that H(2)O(2) induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-phosphorylation-dependent pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H(2)O(2) (100 microM) activated the Akt/eNOS pathway, higher levels (500 microM) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfhydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorylation events induced by 100 microM H(2)O(2). GSH was not a target thiol in this system because buthionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane surface and intracellular free thiols was associated with the inhibition of phosphorylation, suggesting that oxidation of non-GSH thiols inhibits the H(2)O(2)-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols, suggesting a more prominent role for the former. Similarly, DEM and CDNB inhibited TNF-alpha-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS.
... Cells expressing the Cdc25A WT protein, lacking IL-7, had begun to undergo apoptosis and shrink in size, as indicated by decreased FSC, whereas cells expressing Cdc25A-DP remained viable and did not undergo atrophy. Therefore, expression of Cdc25A-DP replaced the IL-7 signal for cycling and cell maintenance for 2–5 d of cytokine withdrawal, after which cells expired likely due to loss of nutrient uptake (Khaled and Durum, 2003). Untransfected cells or cells expressing the WT protein shrink and die after 36–48 h of cytokine deprivation. ...
Lymphocytes are the central mediators of the immune response, requiring cytokines for survival and proliferation. Survival signaling targets the Bcl-2 family of apoptotic mediators, however, the pathway for the cytokine-driven proliferation of lymphocytes is poorly understood. Here we show that cytokine-induced cell cycle progression is not solely dependent on the synthesis of cyclin-dependent kinases (Cdks) or cyclins. Rather, we observe that in lymphocyte cell lines dependent on interleukin-3 or interleukin-7, or primary lymphocytes dependent on interleukin 7, the phosphatase Cdc25A is the critical mediator of proliferation. Withdrawal of IL-7 or IL-3 from dependent lymphocytes activates the stress kinase, p38 MAPK, which phosphorylates Cdc25A, inducing its degradation. As a result, Cdk/cyclin complexes remain phosphorylated and inactive and cells arrest before the induction of apoptosis. Inhibiting p38 MAPK or expressing a mutant Cdc25A, in which the two p38 MAPK target sites, S75 and S123, are altered, renders cells resistant to cytokine withdrawal, restoring the activity of Cdk/cyclin complexes and driving the cell cycle independent of a growth stimulus.
... One additional factor that is vital for the survival of naive CD4 T cells is IL-7. The IL-7 receptor is expressed on resting CD4 and CD8 T cells and is responsible for providing signals that promote survival of the resting cells, such as upregulation of Bcl-2 expression [58]. Importantly, it has been shown that levels of IL-7 in the serum of older humans is significantly reduced compared to younger individuals [59]. ...
Survival and homeostatic division of naive CD4 T cells is regulated by the cellular and non-cellular milieu and together these processes ensure that a population of naive CD4 T cells persists into old age. However, the naive CD4 T cells from aged animals show reduced IL-2 production, proliferation, helper function and effector generation and memory function. We explore here whether the age-related defects in naive CD4 T cells are due to the aged environment from which they come or to intrinsic defects that are caused by homeostasis and their long lifespan.
HIV-1 establishes latency primarily by infecting activated CD4+ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. Here we describe both the original and a simplified version of HIV-1 latency models that mimics this process using replication competent viruses. Our model allows generation of large numbers of latently infected CD4+ T cell to dissect molecular mechanisms of HIV latency and reactivation.
Retinoic acid receptor-related (RAR) orphan receptor γ (Rorγt+) innate lymphoid cells or group 3 innate lymphoid cells (ILC3s) play key roles in the development of lymphoid tissues, maintenance of epithelial tissue homeostasis, and repair and defense against pathogens encountered at the body's barriers. These cells produce interleukin (IL)-22, IL-17A, and lymphotoxin (LT)α1β2 which are critical cytokines necessary for induction of the protective pathways at epithelial and mucosal surfaces. The ILC3 are a heterogeneous group of cells. Significant efforts have been made to decipher the transcriptional program that regulates their development and to understand the functions and mechanisms of actions of the individual subsets in providing immune protection.
Although several pro-inflammatory cytokines including interleukin-6 (IL-6), IL-7, IL-12/IL-23, IL-17, IL-2, interferon, and the anti-inflammatory cytokines, IL-4/IL-13, IL-10, and IL-22, all activate the Janus kinase/signal transducers and activators of transcription (JAK/ STAT) pathway, in autoimmune disorders, a skewing of the cytokine repertoire in favor of pro-inflammatory cytokines results in amplifying the effects of pro-inflammatory cytokines. An apparent deficiency of anti-inflammatory cytokines to counterbalance the 'ramping up' of pro-inflammatory cytokine-mediated activation of JAK/STAT is also significant, while endog-enous negative regulators of cytokine signaling and JAK/STAT activation may also be com-promised. In addition, JAK/STAT pathway activation can result in activation of stress-activated protein/mitogen-activated protein kinase (SAP/MAPK) and phosphatidylinositol-3-kinase/ Akt/ mammalian target of rapamycin pathways that are instrumental in promoting matrix metal-loproteinase gene expression, aberrant cell survival, and osteoclast differentiation. The critical role played by pro-inflammatory cytokines in differentially activating JAK/STAT and parallel signal transduction pathways resulted in the development of several cytokine/cytokine receptor neutralizing monoclonal antibodies and fusion proteins that are currently employed for treating rheumatoid arthritis, Crohn's disease, and psoriasis. Small molecule inhibitors (SMIs) that target specific JAK enzymes have led to the development of CP690550, a JAK3-specific SMI, which is the first JAK-specific SMI to reach phase III in a rheumatoid arthritis clinical trial.
T cell adequate function is critical for defense against pathogens. Transient disruption of T cell homeostasis occurs when primarily naive cells undergo antigen-driven expansion and acquire effector functions. Effector T cells then either undergo programmed cell death (PCD, it occurs usually as massive apoptosis during the contraction phase of the immune response) or survive to become memory cells. Two main pathways of effector T cell PCD are discussed in the review: activation induced cell death (AICD), which is a form of extrinsic apoptosis, and neglect-induced death (NID), which is an intrinsic one. Initial studies using in vitro models supported a role of AICD, mostly initiated by TCR receptor triggering in immune contraction. However, it was not finally supported by either recent in vivo experiments or current review authors' clinical studies concerning primed T cell apoptosis in chronic inflammatory lower airway diseases. Actually, Bcl-2 family members seem to be critical for the culling of T cell responses. The antiapoptotic molecule Bcl-2 and its proapoptotic antagonist Bcl-2, both under upstream control of autocrine interleukin-2, are the most probable candidates for regulators of T cell contraction. Other possible mechanisms regulating the process of contraction such as death receptor ligation, the impact of cytokines, as well as the importance of transcription factor NF-κB, are discussed. Additionally, attention is turned to the potential role of T cell survival/apoptosis regulation in future therapies of some diseases, including tumors and lung fibrosis.
Over the past 13 years, numerous crystal structures of complexes of the common γ-chain (γ(c) ) cytokine receptors and their cytokines have been solved. Even with the remarkable progress in the structural biology of γ(c) receptors and their cytokines or interleukins, there are valuable lessons to be learned from the structural and biophysical studies of interleukin-7 (IL-7) and its α-receptor (IL-7Rα) and comparisons with other γ(c) family members. The structure of the IL-7/IL-7Rα complex teaches that interfaces between the γ(c) interleukins and their receptors can vary in size, polarity, and specificity, and that significant conformational changes might be necessary for complexes of interleukins and their receptors to bind the shared, activating γ(c) receptor. Binding, kinetic, and thermodynamic studies of IL-7 and IL-7Rα show that glycosylation and electrostatics can be important to interactions between interleukins and their receptor, even where the glycans and charged residues are distant from the interface. The structure of the IL-7Rα homodimer is a reminder that often-ignored non-activating complexes likely perform roles just as important to signaling as activating complexes. And last but not least, the structural and biophysical studies help explain and potentially treat the diseases caused by aberrant IL-7 signaling.
In the human acute myeloid leukemia cell line M07e, the growth factor interleukin-3 (IL-3) induces ROS formation, positively affecting Glut1-mediated glucose uptake and cell survival. The effect of IL-3 and exogenous hydrogen peroxide on cell viability seems to be mediated through inhibition of the cell death commitment, as shown by apoptotic markers such as caspase activities, apoptotic nuclei, and changes in the amount of proteins belonging to the Bcl-2 family. The pivotal role of ROS is confirmed using various antioxidants, such as EUK-134, ebselen, TEMPO, and hydroxylamine probe. In fact, these antioxidants, acting through different mechanisms, decrease glucose transport activity and cell proliferation activated by IL-3 or by low concentrations of hydrogen peroxide. Moreover, antioxidants foster programmed cell death commitment, as shown by the cited apoptotic parameters. EUK-134, a combined superoxide dismutase/catalase mimetic, opposes the effects of IL-3 and H2O2, decreasing phosphorylation levels of signaling enzymes such as Akt, Src tyrosine kinase, and ERK. Results show that ROS production induced by IL-3 can protect leukemic cells from apoptosis, the effect being counteracted by antioxidants. This mechanism may play an important role in supporting acute myeloid leukemia treatment, thus representing a novel therapeutic strategy.
The limitations of current anti-retroviral therapies (ART) and the lack of a valid anti-HIV-1 vaccine candidate underscore the need for new therapeutic concepts aiming at the eradication of HIV-1, which represents at the same time an ideal goal and a major challenge for AIDS research. At present, this aim is unattainable due to the existence of cellular and anatomical reservoirs of persistent infection. Memory CD4(+) T cells comprise the largest pool of cells harboring silent, stably integrated HIV-1, which remains undetected by the immune system and refractory to conventional anti-retroviral drugs. The eradication of latent HIV-1 reservoirs will require new, potent and specific therapeutic strategies, which in turn must rely upon a deeper understanding of HIV-1 latency. To facilitate the advancement of our knowledge in this new area of research, several in vitro models of HIV-1 latency in CD4(+) T cells have been established. Here, we dissect and critically compare the rationale behind each experimental approach. Furthermore, we outline new avenues of research that will benefit from these models, including the push toward the development of new classes of viral eradication drugs.
Interleukin-7 (IL-7) is required for T cell development and for maintaining and restoring homeostasis of mature T cells. IL-7 is a limiting resource under normal conditions, but it accumulates during lymphopaenia, leading to increased T cell proliferation. The administration of recombinant human IL-7 to normal or lymphopenic mice, non-human primates and humans results in widespread T cell proliferation, increased T cell numbers, modulation of peripheral T cell subsets and increased T cell receptor repertoire diversity. These effects raise the prospect that IL-7 could mediate therapeutic benefits in several clinical settings. This Review summarizes the biology of IL-7 and the results of its clinical use that are available so far to provide a perspective on the opportunities for clinical application of this cytokine.
The cytokine interleukin-7 (IL-7) has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in the metabolism of glucose that was attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter GLUT-1 and two glycolytic enzymes, hexokinase II (HXKII) and phosphofructokinase-1 (PFK-1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Readdition of IL-7 to cytokine-deprived lymphocytes restored the transcription of the HXKII gene within 2 h, but not that of GLUT-1 or PFK-1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-bromopyruvate or specific small-interfering RNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, whereas overexpression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes.
HIV-1 establishes latency primarily by infecting activated CD4(+) T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. The lack of a valid cellular model to study HIV-1 latency has hindered advances in the understanding of its biology. In this study, we attempted to model HIV-1 latency using human primary CD4(+) T cells infected in vitro with HIV-1 after activation with Ag-loaded dendritic cells and then brought back to quiescence through a resting phase in the presence of IL-7. During the resting phase, expression of cellular activation markers disappeared and cell proliferation and viral replication ceased, but resumed following restimulation of rested cells with Ag or mAbs directed to CD3/CD28. In addition, higher cell death rates were observed in HIV-1-infected than uninfected cultures during secondary but not primary stimulation. Thus, this system may allow us to study the biology of HIV-1 latency, as well as the mechanisms of CD4(+) T cell death following HIV-1 reactivation.
Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-X(L) expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4(+) and CD8(+) T cells. Transplantation of RAG-2-eGFP-transgenic BM revealed that proliferating eGFP(lo)CD44(hi) donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFP(hi)CD44(lo) recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester-labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44(hi) phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.
The impaired elimination of self-reactive T cells is one factor that contributes to autoimmunity. Although the mechanism of thymic negative selection has been studied for decades, recent data demonstrate that the mechanisms underlying this fundamental process remain extremely controversial. Nonetheless, new models such as the Aire-deficient mice have demonstrated the importance of thymic negative selection in autoimmune disease progression in mice and humans.
Lymphocyte populations in the immune system are maintained by a well-organized balance between cellular proliferation, cellular survival and programmed cell death (apoptosis). One of the primary functions of many cytokines is to coordinate these processes. In particular, the interleukin (IL)-2 family of cytokines, which consists of six cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) that all share a common receptor subunit (gammac), plays a major role in promoting and maintaining T lymphocyte populations. The details of the molecular signaling pathways mediated by these cytokines have not been fully elucidated. However, the three major pathways clearly involved include the JAK/STAT, MAPK and phosphatidylinositol 3-kinase (P13K) pathways. The details of these pathways as they apply to the IL-2 family of cytokines is discussed, with a focus on their roles in proliferation and survival signaling.
In the thymus, CD4+ or CD8+ single-positive (SP) thymocytes develop and mature by positive and negative selection or undergo "death by neglect". CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)-null mice. The level ofROS in PrxII-null thymocytes is higher than that in wild-type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII-null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double-positive (DP) thymocytes in thymus of PrxII-null mice is lower than that in wild-type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII-null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII-null thymocytes than in wild-type thymocytes. In the presence of low serum concentrations, PrxII-deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin- or OKT3-stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.
Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1(-/-) mice are developmentally and functionally distinct.
Maintenance of a sufficient population of naive CD8+ T cells in the peripheral lymphoid compartment is critical for immunocompetence. Peripheral T cell number is a function of T cell generation, survival, and death. Homeostasis, a critical balance between survival and death, must exist to prevent either lymphopenia or lymphocytosis. In the current review, we discuss known requirements for the survival of naive peripheral CD8+ T cells as well as mechanisms of death when survival signals are lost. We also discuss associations between survival and homeostasis-driven proliferation, and highlight the gaps in our knowledge of these critical processes.
During an immune response, peripheral T-cell populations expand and then contract as the response subsides, thus maintaining a fairly constant number of CD4 and CD8 T cells throughout the life of the individual. The important factors that control this homeostasis are now beginning to be understood. Interleukin-7 (IL-7) has emerged as a central regulator of the survival and homeostasis of CD4 and CD8 T cells. Both naive and memory T-cell populations are highly dependent on the presence of IL-7 for their persistence and survival. In this Review, we discuss the role of IL-7 in the survival and homeostasis of naive and memory T cells and how that role is regulated by other factors.
Adaptive immunity protects against infection and cancer but is also a potential threat to the host because of the risk of excessive inflammation or the development of autoimmunity and allergy. Therefore, immune responses are subject to negative regulation. An important aspect of negative regulation is the generation of adaptive regulatory T (Treg) cells, which are currently thought to be induced mainly by immature (or partially mature) dendritic cells (DCs). In this Opinion, arguments will be presented for the concept that mature DCs can also have a key role in the development of Treg cells, both under conditions of immunity and homeostasis. Knowledge regarding the biology of regulatory DCs is of crucial importance for the development of novel therapeutic tools to correct immune aberrances in disease.
It has become clear that the regulation of T cell numbers is under peripheral homeostatic control. However, the rules for homeostasis vary with the T cells' differentiation state and the overall T cell number in the animal. Furthermore, homeostatic pressures can cause unexpected changes in T cell differentiation and function which might promote or dampen T cell reactivity. In this review, we focus on the role of peptide/MHC and cytokine interactions in regulating the size and composition of the T cell pool.
Apoptosis of circulating CD8+ T lymphocytes is a frequent finding in patients with cancer. T-cell output by the thymus or antigen-driven expansion of circulating T cells could compensate for apoptosis and thus normalize their homeostasis. We studied the frequency of recent thymic emigrants (RTE) identified by T-cell receptor excision circles (TREC) and of naive and memory T-cell subsets in peripheral blood samples obtained from 39 patients with head and neck cancer (HNC) and 33 age-matched controls (NC). TREC numbers were determined by real-time quantitative PCR, and CD8+CD45RO-CD27+ or CD4+CD45RO-CD27+ T-cell subsets were quantified by flow cytometry. Age-associated decreases in TREC numbers and proportions of naive CD8+ and CD4+ T-cell subsets were significantly greater in cancer patients than NC. In contrast, the memory compartment was expanded, with increased proportions of CD4+CD45RO+ but not CD8+CD45RO+ T cells, in cancer patients vs. NC. These alterations did not normalize in patients who were NED. The data suggest that lower thymic output combined with rapid turnover of naive CD8+ T cells account for altered lymphocyte homeostasis in HNC patients. The defect persists long after curative treatments and may contribute to immune cell dysregulation in patients with cancer.
IL-7 is essential for the development and survival of T lymphocytes. This review is primarily from the perspective of the cell biology of the responding T cell. Beginning with IL-7 receptor structure and regulation, the major signaling pathways appear to be via PI3K and Stat5, although the requirement for either has yet to be verified by published knockout experiments. The proliferation pathway induced by IL-7 differs from conventional growth factors and is primarily through posttranslational regulation of p27, a Cdk inhibitor, and Cdc25a, a Cdk-activating phosphatase. The survival function of IL-7 is largely through maintaining a favorable balance of bcl-2 family members including Bcl-2 itself and Mcl-1 on the positive side, and Bax, Bad and Bim on the negative side. There are also some remarkable metabolic effects of IL-7 withdrawal. Studies of IL-7 receptor signaling have yet to turn up unique pathways, despite the unique requirement for IL-7 in T cell biology. There remain significant questions regarding IL-7 production and the major producing cells have yet to be fully characterized.
Chemokine C receptor 7 (CCR7) expression is important for lymphocyte homing to tissues. We hypothesized that CCR7 also plays a role in CD8(+) T-cell protection from apoptosis. Its expression was determined on circulating T cells in patients with cancer and related to that of molecules responsible for lymphocyte susceptibility/resistance to apoptosis. Peripheral blood mononuclear cells were obtained from 36 patients with squamous cell carcinoma of the head and neck and 16 normal controls. Multicolor flow cytometry was used to evaluate CCR7, Fas, Bax, and Bcl-2 expression in CD8(+) T cells. Annexin V binding to CD8(+)CCR7(+) and CD8(+)CCR7(-) T-cell subsets was compared. Fewer CD8(+)CCR7(+) T cells bound Annexin V than CD8(+)CCR7(-) T cells in normal control and patients (P < 0.0001). CCR7 expression correlated with higher Bcl-2 but lower Bax and Fas expression levels in CD8(+) T cells in both normal control and patients (P < 0.0001). In patients, the CD8(+)CCR7(+) subset was reduced relative to normal control (P = 0.008) and replaced with an excess of apoptosis-sensitive CD8(+)CCR7(-) T cells. To study CCR7 signaling, CD8(+) T cells were stimulated with CCR7 ligands, chemokine C ligands 19 or 21. Ligand binding to CCR7 resulted in phosphorylation of Akt and increased Bcl-2 expression in CD8(+)CCR7(+) T cells, suggesting that CCR7 protects effector T cells from apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. The absence of CCR7 expression on the majority of CD8(+) T cells in the peripheral circulation of patients with squamous cell carcinoma of the head and neck contributes to apoptosis and a rapid turnover of these effector cells.
The balance between life and death of a cell regulates essential developmental processes in multicellular organisms. Apoptotic cell death is a complex, stepwise program involving multiple protein components that trigger and execute the demise of the cell. Of the many triggers of apoptosis, most are not well understood, but some key components have been identified, such as those of the Bcl-2 family, which function as anti-apoptotic or proapoptotic factors. Bax, a pro-apoptotic member of this family, has been shown to serve as a component of many apoptotic triggering cascades and its mechanism of action is the focus of intense study. Herein we discuss current, differing ideas on the function of Bax and its structure, and suggest novel mechanisms for how this death protein targets mitochondria, triggering apoptosis.
Cytokines contribute to T cell homeostasis at all stages of T cell existence. However, the particular cytokine involved varies as T cells progress from a naïve through an activated to a memory state. In many cases the important cytokines are members of the interleukin 2 subfamily of the short-chain type I cytokines. A case is made for the idea that the evolutionary divergence of the short-chain family allowed for concurrent divergence in leukocytes.
C57BL/6 mice genetically deficient in interleukin 15 (IL-15−/− mice) were generated by gene targeting. IL-15−/− mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8+ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these
populations in IL-15−/− mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15−/− mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15
in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed
by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15−/− mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15−/− mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective
response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid
lineages. Moreover, the ability to rescue lymphoid defects in IL-15−/− mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.
Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility,
and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kγ were generated. We show that PI3Kγ controls
thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kγ-deficient
neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein
(G protein)–coupled receptor (GPCR) agonists and chemotactic agents. PI3Kγ links GPCR stimulation to the formation of phosphatidylinositol
3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated
kinases 1 and 2. Thus, PI3Kγ regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.
The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common gamma (gamma c) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in gamma c signaling and lymphoid development.
The activation of Janus protein tyrosine kinases (Jak) and STAT (signal transducer and activator of transcription) proteins has recently been linked to the signal transduction mechanism of several cytokines. IL-7 was observed to induce a rapid and dose-dependent tyrosine phosphorylation of Jak 1 and Jak 3 and concomitantly, the tyrosine phosphorylation and DNA binding activity of multiple STAT proteins. The STAT proteins utilized by IL-7 were identical to those induced by IL-2 and could be identified as various STAT 5 isoforms. Moreover, the induction of both Jak 1 and 3, and STAT 5 activity strongly correlated with the growth-promoting effects of IL-7, suggesting that this signal transduction mechanism may play a key role in IL-7-induced proliferation.
Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.
I have used the transfer of primed lymphocytes into syngeneic irradiated recipients to investigate whether the persistence of antigen is required in the long-term maintenance of cytolytic T cell memory to influenza virus. Animals were immunized with influenza virus (A/WSN) and used 17 wk later as either donors for T cells or as lethally irradiated recipients. Naive age-matched mice served as controls. At intervals of 4, 8, 16, and 25 wk after T cell transfer, experimental and control groups were immunized with a heterologous virus (A/JAP) and splenocytes tested for lytic activity to influenza virus 3 and 6 d after immunization. Lytic activity 3 d after infection (a property exclusive to a memory cytotoxic T cell response) (Effros, R. B., J. Bennink, and P. C. Doherty. 1978. Cell. Immunol. 36:345.; and Hill, A. B., R. V. Blanden, C. R. Parrish, and A. Müllbacher. 1992. Immunol. Cell Biol. 70:259), was only observed by primed and naive irradiated recipients reconstituted with memory T cells. No day 3 responses were observed when naive T cells were transferred into irradiated primed or unprimed recipients. These observations demonstrate that cytolytic T cell memory to influenza virus is long lived in the absence of antigen.
The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.
A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B- and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8- T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the major cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous injection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 injection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.
We have investigated cell metabolism during apoptosis in the murine interleukin-3 (IL-3)-dependent cell line Bo and two derivative clones (B14 and B15) overexpressing human bcl-2a. On removal of IL-3, Bo cells underwent apoptosis within 8 h, whereas B14 and B15 cells were resistant for at least 24 h. Metabolically, Bo, B14, and B15 cells were indistinguishable from each other. All were insensitive to mitochondrial poisons, derived ATP entirely by glycolysis, and maintained similar mitochondrial membrane potentials measured by rhodamine-123 fluorescence with or without IL-3. All virtually ceased glycolysis and production of lactic acid on IL-3 withdrawal but maintained intracellular [ATP] until in Bo cultures the cells began to apoptose. B14 and B15 cells became glycolytically arrested but maintained stable ATP levels during protection from apoptosis. Depletion of intracellular ATP by uncoupling the mitochondrial ATPase with 2,4-dinitrophenol or carbonyl cyanide p-trifluoromethoxyphenylhydrazone induced apoptosis in Bo cells with or without IL-3, but not in B14 or B15 cells. bcl-2-overexpressing cells were recoverable with high plating efficiency even after prolonged exposure to 2,4-dinitrophenol. We conclude that IL-3 withdrawal leads to arrest of energy metabolism in which ATP levels are maintained. In Bo cells this is followed by apoptosis, whereas in bcl-2-overexpressing cells this state is stably prolonged. ATP depletion is a strong apoptotic signal which overrides IL-3 signaling in normal cells but is ineffective in bcl-2-overexpressing cells. Prolonged metabolic arrest and resistance to ATP depletion facilitated by bcl-2 are both reversible. Persistent reversible metabolic dormancy would provide cells with a survival advantage in nonsustainable environments (e.g. hypoxia or substrate lack) and suggests a mechanism for the survival advantage displayed by cells overexpressing bcl-2.
Members of the Bcl-2 family (including Bcl-2, Bcl-XL, and Bax) play key roles in the regulation of apoptosis. These proteins are believed to be membrane-associated and have been
proposed to regulate apoptosis through both homodimerization and heterodimerization. We have found that whereas Bcl-2 is predominantly
membrane-associated as previously reported, significant amounts of Bcl-XL and most of the Bax proteins are not membrane-associated and thus appear in the cytosolic fraction of thymocyte and splenocyte
extracts. This finding allows the study of the dimerization properties and conformation of these proteins in the absence of
detergent perturbation. For this analysis, we have produced monoclonal antibodies that are specific for known epitopes of
Bax, Bcl-2, and Bcl-XL. An antibody to an N-terminal epitope (α uBax 6A7) between amino acids 12 and 24 fails to bind the soluble cytosolic form
of Bax, indicating that this epitope is normally buried. Nonionic detergents alter the Bax conformation to expose this epitope.
In the presence of nonionic detergent, the 6A7 antibody avidly binds the monomeric form of Bax, but not Bax complexed with
either Bcl-XL or Bcl-2. In contrast, a monoclonal antibody to an adjacent epitope of Bax (α mBax 5B7) within amino acids 3–16 binds the
soluble and detergent-altered forms of Bax and also binds the Bax·Bcl-XL or the Bax·Bcl-2 complex. Surprisingly, in the absence of detergent Bax fails to form homodimers or heterodimers with Bcl-XL. These results demonstrate a novel conformational state of members of the Bcl-2 family under a physiological condition that
is distinct from the detergent-altered state that forms dimers and is currently believed to regulate apoptosis.
Mice lacking functional IL-7 or IL-7R alpha genes are severely deficient in developing thymocytes, T cells, and B cells. IL-7 and IL-7 receptor functions are believed to result in lymphoid cell proliferation and cell maturation, implying signal transduction pathways directly involved in mitogenesis and elaboration of developmentally specific new gene programs. Here, we show that enforced expression of the bcl-2 gene in T-lymphoid cells (by crossing in the Emu-bcl-2 transgene) in IL-7R alpha-deficient mice results in a significant restoration of thymic positive selection and T cell numbers and function. We propose cell survival signals to be the principal function of IL-7R engagement in thymic and T cell development.
To ensure that the mature T cell repertoire is MHC-restricted yet not autoreactive, cortical thymocytes that express low levels of the TCR/CD3 complex along with CD4 and CD8 (double positive cells) are subjected to positive and negative selection. Surviving cells lose either CD4 or CD8 (single positive cells) and are located primarily in the thymic medulla. bcl-2, a novel proto-oncogene that promotes cell survival by inhibiting programmed cell death (apoptosis), may be an important protein in regulating cell survival during thymocyte development. We have examined the expression of bcl-2 during T cell development by using human thymocytes. Consistent with previous studies, human thymic tissue sections stained for bcl-2 revealed occasional bcl-2+ cells within the thymic cortex and intense staining of virtually all medullary thymocytes. More quantitative western blot analysis and S1 nuclease protection assay revealed that single positive thymocytes contained approximately 2 to 3 times the level of bcl-2 protein and 3 to 4 times the level of bcl-2 mRNA as double positive thymocytes. Flow cytometric analysis of purified double positive thymocytes revealed that minimal amounts of bcl-2 protein was in fact detectable in most cells, although a small subpopulation (10-20%) contained higher levels. In contrast, brighter staining for bcl-2 was observed in virtually all single positive thymocytes. Surprisingly, CD4-CD8- thymocytes (both CD3- and CD3+) expressed the same amount of bcl-2 as did the single positive thymocytes. Because a large percentage of CD3-CD4-CD8- cells are cycling, we examined the effect of mitogenic stimulation on bcl-2 expression by double positive thymocytes by using western blot analysis. bcl-2 expression in double positive thymocytes could not be induced by cell cycle entry following stimulation with PMA and ionomycin. Our data demonstrate that bcl-2 expression is biphasic during T cell development. Both CD3-CD4-CD8- and CD3+CD4+ and CD3+CD8+ thymocytes express high levels of bcl-2. Therefore, diminished bcl-2 expression in double positive thymocytes seems to be the result of specific down-regulation in order to facilitate the selection CD4+CD8+ thymocytes.
Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.
The balance between life and death of a cell regulates essential developmental processes in multicellular organisms. Apoptotic cell death is a complex, stepwise program involving multiple protein components that trigger and execute the demise of the cell. Of the many triggers of apoptosis, most are not well understood, but some key components have been identified, such as those of the Bcl-2 family, which function as anti-apoptotic or pro-apoptotic factors. Bax, a pro-apoptotic member of this family, has been shown to serve as a component of many apoptotic triggering cascades and its mechanism of action is the focus of intense study. Herein we discuss current, differing ideas on the function of Bax and its structure, and suggest novel mechanisms for how this death protein targets mitochondria, triggering apoptosis.
The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.
Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
The importance of Jak-Stat pathway signaling in regulating cytokine-dependent gene expression and cellular development/survival is well established. Nevertheless, advances continue to be made in defining Jak-Stat pathway effects on different cellular processes and in different organisms. This review focuses on recent advances in the field and highlights some of the most active areas of Jak-Stat pathway research.
IL-7 receptor-deficient (IL-7R ‐/‐ ) mice are lymphopenic as a result of defective cell production at early steps in both B and T lymphopoiesis. In the bone marrow, there is an incomplete block in B cell development at the transition from the pro-B to the pre-B cell stage. As a consequence, peripheral lymphoid organs of IL-7R ‐/‐ mice contain abnormally low numbers of mature surface (s) Ig-expressing B cells and this is accompanied by a relative increase in immature sIg ‐ B cells. Transgenic expression of the anti-apoptotic protein Bcl-2 in IL-7R ‐/‐ mice rescues the defect in T cell development and in mature T cell function. The present report shows that constitutive expression of Bcl-2 is incapable of rescuing B lymphopoiesis in IL-7R ‐/‐ mice but can enhance survival of those mature B cells which escape the developmental arrest. Thus the essential role of IL-7R signaling in B lymphoid cells cannot be replaced by Bcl-2, indicating that in B lymphopoiesis IL-7R signaling is necessary for promoting cell division and/or for inhibiting a Bcl-2-insensitive pathway to apoptosis.
T-cell receptor (TCR) genes need to be rearranged by a site specific-VDJ recombinase before they are expressed. This process, initiated in CD44+25+ thymocytes, takes place during the early stages of T-cell differentiation in the thymus. Interleukin-7 receptor α chain knockout (IL-7R−/−) mice are severely deficient in B-lymphocytes and αβ T-cells and completely lack the γδ T-cell lineage. Thymocyte development is arrested at a very early stage (DN CD44+CD25−). Because this arrest is earlier than in mice with a block in VDJ recombination, we examined the rearrangement status of TCR genes in thymocytes from IL-7R−/− mice. The TCRβ locus showed a nearly normal pattern of VDJ rearrangements, consistent with the presence of αβ T-cells in these mice. However, TCRγ locus rearrangement was absent or severely reduced for all the Vγ genes analyzed (Vγ3, Vγ4, Vγ1.1, Vγ1.2 and Vγ2). In contrast, the δ locus showed little reduction in rearrangement. The defect in γ rearrangements in IL-7R−/− thymocytes is not simply due to an absence of mature γδ T-cells, since TCRδ−/− mice, which also have only αβ T-cells, had normal levels of γ and δ rearrangements. These findings indicate that one or both of the two known ligands of IL-7R, IL-7 and thymic stromal lymphopoietin (TSLP) serves as an extrinsic signal to specifically rearrange the TCRγ locus.
A central paradigm of T cell development is that CD4+8+ (DP) thymocytes differentiate into CD4+ or CD8+ T cells in response to intrathymic signals that extinguish transcription of the inappropriate coreceptor molecule. Contrary to this prevailing paradigm, we now demonstrate that signaled DP thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Remarkably, thymocytes that have selectively terminated CD8 transcription can be signaled by IL-7 to differentiate into CD8+ T cells by silencing CD4 transcription and reinitiating CD8 transcription, events we refer to as “coreceptor reversal.” These observations significantly alter our understanding of CD8+ T cell differentiation and lead to a new perspective (“kinetic signaling”) on CD4/CD8 lineage determination in the thymus. These observations also suggest a novel mechanism by which bipotential cells throughout development can determine their appropriate cell fate.
The requisite molecular interactions for CD8 T cell memory were determined by comparison of monoclonal naı̈ve and memory CD8+ T cells bearing the T cell receptor (TCR) for the HY antigen. Naı̈ve T cells required only the right major histocompatibility
complex (MHC) class I–restricting molecule to survive; to expand, they also needed antigen. In contrast, for survival, memory
cells did not require the restricting MHC allele, but needed only a nonspecific class I; for expansion the correct class I,
but not antigen, was required. Thus, maintenance of CD8 T cell memory still required TCR–MHC class I interactions, but memory
T cells may have a lower functional activation threshold that facilitates secondary responses.
Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.
The vast majority of cortical thymocytes die during T cell development while those that survive this selective process accumulate in the medulla. bcl-2, an inner mitochondrial membrane protein, has been shown to inhibit apoptosis in certain cell lines. In the thymus, bcl-2 is regionally localized to the mature T cells of the medulla. To assess the role of bcl-2 in the programmed death of thymocytes, we generated transgenic mice that redirected bcl-2 expression to cortical thymocytes. bcl-2 protected immature CD4+8+ thymocytes from glucocorticoid, radiation, and anti-CD3-induced apoptosis. Moreover, bcl-2 altered T cell maturation, resulting in increased percentages of CD3hi and CD4-8+ thymocytes. Despite this, clonal deletion of T cells that recognize endogenous superantigens still occurred. This transgenic model indicates that multiple death pathways operate within the thymus that can be distinguished by their dependence on bcl-2.
Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.
Haemopoietic cell development in vivo occurs in restricted sites in association with stromal cells. Haemopoiesis in vitro can be induced in the absence of stromal cells, provided the haemopoietic cells are supplied with appropriate growth stimulatory molecules. Evidence indicates that the same, or functionally similar, growth factors are normally supplied in vivo by the surrounding stromal cells and that the control of haemopoietic cell proliferation and development is regulated locally and is mediated by cell-cell interactions. We have been studying the effects of a growth factor which induces self-renewal and differentiation of multipotential stem cells as well as proliferation and development of lineage-restricted progenitor cells and activation of mature cells. Because of the wide range of activities embraced by this molecule we have termed it haemopoietic cell growth factor (HCGF). It is also known as interleukin 3 and multi-CSF. HCGF allows the survival, proliferation and development of cells and can be used to generate continuously growing, non-leukaemic, factor-dependent cell lines, in vitro (FDC-P). In the absence of HCGF. FDC-P cells die within hours. We have shown that HCGF may exert its primary effects (in terms of cell survival) on ATP generation, via its influence on glucose transport. Studies are also described which indicate that a primary event in differentiation induced by HCGF involves ADP-ribosylation of membrane-associated proteins. The significance of these findings for normal haemopoiesis and in leukaemogenesis is discussed.
Memory is a hallmark of the immune system and ever since its recognition there has been considerable interest in understanding how immunity is maintained. The current model is that long-term memory is dependent on persistent antigenic stimulation. We report here results that challenge this view and provide evidence that antigen is not essential for the maintenance of CD8+ T-cell memory. We show that memory CD8+ cytotoxic T lymphocytes persist indefinitely in the absence of priming antigen, retain the memory phenotype (CD44hi), and provide protection against virus challenge. These findings suggest a re-evaluation of our current thinking on mechanisms involved in maintaining immunity and have implications towards designing effective vaccination strategies.
Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI-3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.
BAX, a heterodimeric partner of BCL2, counters BCL2 and promotes apoptosis in gain-of-function experiments. A Bax knockout mouse was generated that proved viable but displayed lineage-specific aberrations in cell death. Thymocytes and
B cells in this mouse displayed hyperplasia, and Bax-deficient ovaries contained unusual atretic follicles with excess granulosa cells. In contrast, Bax-deficient males were infertile as a result of disordered seminiferous tubules with an accumulation of atypical premeiotic
germ cells, but no mature haploid sperm. Multinucleated giant cells and dysplastic cells accompanied massive cell death. Thus,
the loss of Bax results in hyperplasia or hypoplasia, depending on the cellular context.
To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and Stat5 in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2R beta and IL-7R that can serve as docking sites for Stat proteins. IL-13 Induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.
It is known that phorbol esters can protect IL-2-dependent lymphocytes against apoptosis induced by IL-2 withdrawal. However, the mechanism of this effect remains unclear. In this article we show that apoptosis induced by IL-2 withdrawal in the CTLL-2 cell line correlates with a decrease in intracellular pH (pHi). Supplementing the incubation medium with phorbol esters during IL-2 deprivation protects CTLL-2 cells against both apoptosis and intracellular acidification. Interestingly, IL-4 also supports short-term cell survival and maintenance of normal pHi. The protein kinase inhibitor staurosporine prevents the protective effects of IL-2, PMA, and IL-4 on apoptosis and intracellular acidification. In contrast, inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride reverts the protective effects of PMA and IL-4, but only weakly affects IL-2-mediated suppression of apoptosis. Taken together, these results indicate that intracellular acidification may be an important event during apoptosis induced by IL-2 deprivation in the CTLL-2 cell line. Moreover, they suggest a key role for protein kinase C activation both in the maintenance of pHi and in the suppression of apoptosis, through mechanisms which rely on the activation of the Na+/H+ antiporter to a different extent, depending on the rescuing factor employed.
To ensure that the mature T cell repertoire is MHC-restricted yet not autoreactive, cortical thymocytes that express low levels of the TCR/CD3 complex along with CD4 and CD8 (double positive cells) are subjected to positive and negative selection. Surviving cells lose either CD4 or CD8 (single positive cells) and are located primarily in the thymic medulla. bcl-2, a novel proto-oncogene that promotes cell survival by inhibiting programmed cell death (apoptosis), may be an important protein in regulating cell survival during thymocyte development. We have examined the expression of bcl-2 during T cell development by using human thymocytes. Consistent with previous studies, human thymic tissue sections stained for bcl-2 revealed occasional bcl-2+ cells within the thymic cortex and intense staining of virtually all medullary thymocytes. More quantitative western blot analysis and S1 nuclease protection assay revealed that single positive thymocytes contained approximately 2 to 3 times the level of bcl-2 protein and 3 to 4 times the level of bcl-2 mRNA as double positive thymocytes. Flow cytometric analysis of purified double positive thymocytes revealed that minimal amounts of bcl-2 protein was in fact detectable in most cells, although a small subpopulation (10-20%) contained higher levels. In contrast, brighter staining for bcl-2 was observed in virtually all single positive thymocytes. Surprisingly, CD4-CD8- thymocytes (both CD3- and CD3+) expressed the same amount of bcl-2 as did the single positive thymocytes. Because a large percentage of CD3-CD4-CD8- cells are cycling, we examined the effect of mitogenic stimulation on bcl-2 expression by double positive thymocytes by using western blot analysis. bcl-2 expression in double positive thymocytes could not be induced by cell cycle entry following stimulation with PMA and ionomycin. Our data demonstrate that bcl-2 expression is biphasic during T cell development. Both CD3-CD4-CD8- and CD3+CD4+ and CD3+CD8+ thymocytes express high levels of bcl-2. Therefore, diminished bcl-2 expression in double positive thymocytes seems to be the result of specific down-regulation in order to facilitate the selection CD4+CD8+ thymocytes.
Bcl-2 protein is able to repress a number of apoptotic death programs. To investigate the mechanism of Bcl-2's effect, we examined whether Bcl-2 interacted with other proteins. We identified an associated 21 kd protein partner, Bax, that has extensive amino acid homology with Bcl-2, focused within highly conserved domains I and II. Bax is encoded by six exons and demonstrates a complex pattern of alternative RNA splicing that predicts a 21 kd membrane (alpha) and two forms of cytosolic protein (beta and gamma). Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. Overexpressed Bax accelerates apoptotic death induced by cytokine deprivation in an IL-3-dependent cell line. Overexpressed Bax also counters the death repressor activity of Bcl-2. These data suggest a model in which the ratio of Bcl-2 to Bax determines survival or death following an apoptotic stimulus.
bcl-2-/-mice complete embryonic development, but display growth retardation and early mortality postnatally. Hematopoiesis including lymphocyte differentiation is initially normal, but thymus and spleen undergo massive apoptotic involution. Thymocytes require an apoptotic signal to manifest accelerated cell death. Renal failure results from severe polycystic kidney disease characterized by dilated proximal and distal tubular segments and hyperproliferation of epithelium and interstitium. bcl-2-/-mice turn gray with the second hair follicle cycle, implicating a defect in redox-regulated melanin synthesis. The abnormalities in these loss of function mice argue that Bcl-2 is a death repressor molecule functioning in an antioxidant pathway.
Interleukin-7 (IL-7) stimulates the proliferation of normal and leukemic B and T cell precursors and T lymphocytes. Activation of the JAK/STAT pathway has been implicated in IL-7R signaling. We investigated which STAT complexes are formed upon stimulation of B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells with IL-7. Gel retardation assays with STAT-binding oligonucleotides showed that IL-7 induces the formation of two major STAT complexes in BCP-ALL cells. Supershifts with anti-STAT antibodies identified these as STAT1 and STAT5 complexes. This pattern of STAT activation was seen in all BCP-ALL cases that respond to IL-7 in proliferation assays. IL-7 also induced STAT/DNA binding in BCP-ALL cases that failed to proliferate in response to IL-7, suggesting that the ability of IL-7R to activate the JAK/STAT pathway per se is not sufficient for proliferation induction. To determine the contribution of the cytoplasmic domain of the IL-7 receptor alpha chain (IL-7R alpha) to activation of STAT proteins, transfectants of the murine pro-B cell line BAF3 were made that express chimeric receptors consisting of the extracellular domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) and the transmembrane and intracellular domains of human IL-7R alpha. Activation of the chimeric G-CSF-R/IL-7R alpha with G-CSF resulted in a full proliferative response and induced the phosphorylation of JAK1 but not JAK2. Major STAT complexes activated by G-CSF-R/IL-7R alpha contained STAT1 or STAT5, while some formation of STAT3-containing complexes was also seen. These findings establish that STAT1 and STAT5, and possibly STAT3, are activated upon stimulation of precursor B cells with IL-7. The data further indicate that the IL-7R alpha chains are directly involved in the activation of JAKs and STATs and have a major role in proliferative signaling in precursor B cells.
Jak3 is a tyrosine kinase mediating cytokine receptor signaling through the association with the common gamma chain of the cytokine receptors such as IL-2, IL-4, IL-7, IL-9, and IL-15. Unlike other members of the Jak family, the expression of Jak3 is highly restricted in hematopoietic cells. To elucidate in vivo function of Jak3, Jak3-deficient mice were generated by homologous recombination. Mice homozygous for Jak3 null mutation showed severe defects, specifically in lymphoid cells. B cell precursors in bone marrow, thymocytes, and both T and B cells in the spleen drastically decreased, although these defects were significantly recovered as aging occurred. Peripheral lymph nodes, NK cells, dendritic epidermal T cells, and intestinal intraepithelial gamma delta T cells were absent. Normal number of hematopoietic stem cells in bone marrow from Jak3-deficient mice and the similar capability to generate myeloid and erythroid colonies as wild-type mice indicated specific defects in lymphoid stem cells. Furthermore, the abnormal architecture of lymphoid organs suggested the involvement of Jak3 in the function of epithelial cells. T cells developed in the mutant mice did not respond to either IL-2, IL-4, or IL-7. These findings establish the crucial role of Jak3 in the development of lymphoid cells.
While early thymic T cell precursor populations and their maturational sequence have been recently identified, the signals driving differentiation are unknown. While cytokines may play an integral role in T cell development, various mouse models rendered genetically deficient for specific cytokines do not display abnormalities in T cell development. Recently, we have generated IL-7 -/- mice and reported that IL-7 plays a unique and nonredundant role in lymphopoiesis. These mice displayed a 10- to 20-fold reduction in the total number of T and B cells. Here, we show that IL-7 -/- mice display a sharp reduction in both the frequency and absolute number of adult thymic gamma delta T cells while retaining normal frequencies of alpha beta T cells. This defect in gamma delta T cell production extends to peripheral organs as IL-7 -/- mice are essentially devoid of splenic and intestinal intraepithelial gamma delta T cells. This aberrant phenotype was traced back to impaired fetal gamma delta T cell maturation. In the absence of IL-7, differentiation of immature V gamma 3 low-CD24+ fetal T cells to mature V gamma 3 high CD24- cells is inhibited. In contrast, NK cell maturation appears to be only mildly affected in the absence of IL-7. To further clarify the role of IL-7 in thymic development, detailed analysis of CD3-4-8- thymic precursors was performed. A partial inhibition in the differentiation of CD44+25+ pro-T cells into CD44-25+ pre-T cells was observed. Unexpectedly, the lack of IL-7 resulted in decreased expression of CD117 (c-kit) on both CD4 low and pro-T cells, suggesting that IL-7 may influence the expression of other cytokine receptors involved in early hemopoietic development. Together, these data clarify the developmental abnormalities during T cell development due to the absence of IL-7.
The protein product of the proto-oncogene bcl-2, originally discovered by virtue of its chromosomal translocation in human follicular centre B cell lymphoma, is a physiological inhibitor of programmed cell death, apoptosis. Initial studies in transgenic mice overexpressing Bcl-2 in B or T lymphocytes demonstrated that Bcl-2 can potently antagonise cell death induced by multiple independent signal transduction routes and can contribute to oncogenesis, particularly in combination with other oncogenes, like c-myc, that promote cell proliferation. Further investigations using crosses between bcl-2 transgenic mice and T cell receptor or immunoglobulin transgenic mice or mutant mice deficient in proper antigen receptor gene rearrangement demonstrated that Bcl-2 can only block death of cells that failed to receive a positive stimulus, "death by neglect', but not activation induced apoptosis. Collectively, these results provide evidence that distinct signalling pathways for apoptosis converge upon a common effector machinery where Bcl-2 acts as an antagonist, but that there also exists a mechanism that can either bypass the Bcl-2 checkpoint or override its protective function. These experimental data are reviewed here and discussed in context of current knowledge of lymphocyte differentiation, tumorigenesis and cell death regulation.
Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.
Interleukin-7 (IL-7) is required for normal lymphoid development. The rearrangement of receptor genes is induced in lymphoid progenitors during their period of IL-7 dependency. The question addressed in this review is: to what extent does IL-7 control the gene rearrangement process? There are two principal types of controls considered. The first is the 'trophic' effect: IL-7 maintains the viability of cells during gene rearrangement, whereas the signal to induce gene rearrangements may be distinct from IL-7. The second possible control is 'mechanistic': IL-7 delivers signals to induce gene rearrangement as well as maintains cell viability. This review covers current thought on how gene rearrangement is regulated and evaluates recent evidence for a mechanistic action of the IL-7 receptor in gene rearrangement.
The requisite molecular interactions for CD8 T cell memory were determined by comparison of monoclonal naïve and memory CD8(+) T cells bearing the T cell receptor (TCR) for the HY antigen. Naïve T cells required only the right major histocompatibility complex (MHC) class I-restricting molecule to survive; to expand, they also needed antigen. In contrast, for survival, memory cells did not require the restricting MHC allele, but needed only a nonspecific class I; for expansion the correct class I, but not antigen, was required. Thus, maintenance of CD8 T cell memory still required TCR-MHC class I interactions, but memory T cells may have a lower functional activation threshold that facilitates secondary responses.
Signals from cytokine and antigen receptors play crucial roles during lymphocyte development. Mice lacking interleukin-7 receptor are lymphopenic, due to a defect in cell expansion at an early stage of differentiation, and the few mature T cells that develop in IL-7R-/- animals are functionally impaired. Both defects were rescued completely by overexpression of the anti-apoptosis protein Bcl-2. T cell progenitors lacking antigen receptor molecules are also blocked in differentiation and die, presumably because they fail to receive a positive signal via their pre-T cell receptor. Surprisingly, Bcl-2 did not promote survival or differentiation of T cells in rag-1-/- mice. These results provide evidence that blocking apoptosis is the essential function of IL-7R during differentiation and activation of T lymphocytes and that pre-TCR signaling blocks a pathway to apoptosis that is insensitive to Bcl-2.
T-cell receptor (TCR) genes need to be rearranged by a site specific-VDJ recombinase before they are expressed. This process, initiated in CD44+25+ thymocytes, takes place during the early stage of T-cell differentiation in the thymus. Interleukin-7 receptor alpha chain knockout (IL-7R-/-) mice are severely deficient in B-lymphocytes and alpha beta T-cells and completely lack the gamma delta T-cell lineage. Thymocyte development is arrested at a very early stage (DN CD44+CD25-). Because this arrest is earlier than in mice with a block in VDJ recombination, we examined the rearrangement status of TCR genes in thymocytes from IL-7R-/- mice. The TCR beta locus showed a nearly normal pattern of VDJ rearrangements, consistent with the presence of alpha beta T-cells in these mice. However, TCR gamma locus rearrangement was absent or severely reduced for all the V gamma genes analyzed (V gamma 3, V gamma 4, V gamma 1.1, V gamma 1.2 and V gamma 2). In contrast, the delta locus showed little reduction in rearrangement. The defect in gamma rearrangements in IL-7R-/- thymocytes is not simply due to an absence of mature gamma delta T-cells, since TCR delta-/- mice, which also have only alpha beta T-cells, had normal levels of gamma and delta rearrangements. These findings indicate that one or both of the two known ligands of IL-7R, IL-7 and thymic stromal lymphopoietin (TSLP) serves as an extrinsic signal to specifically rearrange the TCR gamma locus.
Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. However, the mechanism by which Akt functions to promote survival is not understood. We show that growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery.